RNA interference refers to the phenomenon of the presence of double stranded RNA in a cell eliminating the expression of a gene having the same sequence, while leaving the expression of other unrelated genes undisturbed. This phenomenon, also known as “post transcriptional gene silencing” or “RNA silencing” has been noted in plants for some time, but has only more recently been recognized in animals. Fire et al., Nature, 391, 806 (1998). The discovery of this functionality suggests the possibility of powerful research tools for stopping the production of a specific protein and gene-specific therapeutics operating by this mechanism.
Details of the RNA interference (RNAi) mechanism have recently been elucidated. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme known to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as small interfering RNAs (siRNA) (Berstein et al., Nature, 409, 363 (2001)). Small interfering RNAs derived from Dicer activity are typically about 21-23 nucleotides in length. The RNAi response also features an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC). The RISC complex mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Elbashir et al., Genes Dev., 15, 188 (2001).
One potential impediment to harnessing the RNAi phenomenon in mammalian cells is that the presence of long dsRNAs in these cells also stimulates an interferon response that results in non-specific cleavage of mRNA by a ribonuclease. However, it has been shown that chemically synthesized 21-meric small interfering RNAs (siRNAs) effectively suppress gene expression in several human cell lines without eliciting an interferon response. Elbashir et al., Nature, 411, 494 (2001). In particular, synthetic siRNAs have been found to be most active when containing 21 nucleotide duplexes with two TT nucleotide 3′-overhangs. Elbashir et al., EMBO J., 20, 6877 (2001).
SiRNA's characteristics of high specificity, resistance to ribonucleases, non-immunogenicity and potency suggest tremendous potential as a cell transfection agent for research and therapeutic applications. A variety of strategies exist for delivery of nucleic acid compositions to cells. However, technical difficulties have been encountered in transfecting siRNA into cells. Viral vectors provide relatively efficient delivery, but in some cases present safety problems due to the risk of immunological complications or unwanted propagation in the subject. Adenoviral vectors have shown certain advantages in that they do not integrate into the genome of the cell and can be transduced into resting cells. However, all of these vectors must be prepared by time-consuming recombinant DNA techniques. Oligonucleotides may also be delivered to cells via chemical transfection agents, which have been the subject of much recent work. These agents include polycationic molecules, such as polylysine, and cationic lipids. The liposomal composition Lipofectin® (Felgner et al., PNAS 84:7413, 1987), containing the cationic lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and the neutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widely used. Other methods, such as calcium phosphate mediated transfection, can be used to deliver the oligonucleotides to cells according to reported procedures. However, there is a need for effective, nontoxic siRNA transfection agents that are easy to use and applicable to many cell types.